There is a profound need for regeneration strategies due to trauma and various musculoskeletal diseases. Human pluripotent stem cells (hPSCs) offer a promising tool for regenerative therapies because of their unique capability to self-renew, proliferate nearly indefinitely, and rise to various cell types. In the regeneration process, the temporary extracellular matrix (ECM) provides multiple signals to hPSCs and guides the process of new matrix formation. Fundamental questions on how hPSCs sense and respond to the cues from surrounding environment await further exploration. A main hurdle facing the studies of hPSC-ECM interactions lies in limitation of hPSCs culture techniques which use clumps of cells from dissected colonies. In these culture methods, hPSCs unavoidably pass through the stage of spontaneous differentiation to all three embryonic germ layers before the generation of the desired tissue-specific cell type. The goal of this study is to generate and characterize a monolayer single-cell culture model that allows for the investigation of biological phenomena associated with hPSC lineage specification. We will evaluate the potential of hPSCs cultured in monolayers to undergo epithelial-to-mesenchymal transition (EMT) and differentiate to specific germ layers. Using the single-cell monolayer culture method, we will explore the role of symmetric/asymmetric cell division in regulation of hPSC pluripotency and lineage specification. This will be accomplished by employing microcontact printing techniques to control cell polarity and cytoskeletal organization of at a single-cell level. This project has wo specific aims: (1) determine the potential of hPSCs cultured in a monolayer to undergo EMT, and (2) determine the role of asymmetric division in fate specification of hPSCs. The novel approach developed in this study has broad applicability to many research areas including tissue engineering and regenerative medicine, disease modeling and drug testing.